Natural Antibodies Directed Against Murine Lymphosarcoma Cells Marco A. Pierotti and Ma,ria I. Colnaghi

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SUMMARY-Natural antibodies reacting in a test of complement-dependent cytotoxicity with untreated murine lymphosarcoma cells of thymic origin were found in murine sera. Normal thymus cells were unaffected and unable to absorb the serum activity. The natural antibodies were IgM-like and stable at 56° C. They were not uniformly distributed in the studied strains, and high (C3H/He and C3Hf), intermediate (AKR and CBA/J), and low level strains (BALB/c, DBA/2, C57BL, and C57BL/6J) were found. Hybrids between a high (C3Hf) and a low level strain (C57BL) had the same response as the parental C3Hf mice. An inverse relationship was demonstrated between cytotoxicity of, and susceptibility to, serum of lymphoma cells in a given strain, which suggested that an immunologic modulation was at work. Embryonic cells absorbed the cytotoxic activity of the normal serum.-J Natl Cancer Inst 55: 945949,1975.

Serologic studies on mice demonstrated the presence of natural antibodies against autologous and allogeneic thymus cells (1, 2), endogenous type-C viruses (3-5), cryptic antigen(s) uncovered by neuraminidase (Nase) treatment on autologous, syngeneic, and allogeneic normal lymphoid cells (6), and recently also against tumor cells (7). During the experiments on the characterization of tumor-associated antigens demonstrating the presence of embryonic-type structures on murine lymphoma cells (8, 9), we found, in murine s~ra, natural antibodies reacting with untreated lymphosarcoma cells and not affecting normal lymphoid cells. We investigated their characteristics and strain distribution and whether embryonic antigens interacted with them. MATERIALS AND METHODS

Mice.-Inbred C57BL, C57BL/6J (B6), AKR, BALB/c, C3H/He, and C3Hf strains maintained for several years in this laboratory were used. The C3Hf strain was obtained in 1962 by cesarean section from a C3H/He female; foster nursing was by C57BL females. Sera were also harvested from CBA/J and DBA/2 mice provided by Dr. L. Chieco-Bianchi (Laboratory of Experimental Oncology, University of Padova, Padova, Italy). Tumors.-Most tumors were thymic lymphosarcomas originally induced in this laboratory (8) by 7,12-dimethylbenz[a]anthracene (DMBA) in B6 (B6LyDMBA 1, 2), or by urethan (Ur) in C57BL (C57LyUr23, 24, 31, 32,) C3Hf (C3LyUr8, 11, 12), and BALB/c mice (BALyUrl), and kept in a subcutaneous transplant in syngeneic mice of the same sex as the tumor donors. In addition, 3 transplanted B6 lymphomas, i.e., the G virusinduced E cJ G2 (10), the chemically induced EL 4 (11), and the radiation-induced ERLD (12), were also used. Sera.-Three pools were obtained by collection of sera from the retro-orbital plexus of 2 groups of 80 adult C3Hf and C57BL males bearing a subcutaneous transplant of a syngeneic lymphoma (C3LyUrIl, and 12, and C57LyUr24) and of 1 group of 140 normal 2- to 3month-old C3Hf males. Pools of sera were also obtained

from small groups of 3-month-old normal females of C3H/He, C3Hf, AKR, CBA/J, BALB/c, C57BL, B6, and DBA/2 strains. Antisera to embryo tissue produced by immunizing male C57BL mice with mitomycin Cblocked cells of 10- to 14-day C3Hf embryos [characteristics have been detailed in (9, 13)], were also used. Sera were stored at - 30° C until tested and were not inactivated before use, unless specified. A 2-ml aliquot of the C3Hf normal serum was chromatographed on a Sephadex G-200 gel filtration column (2.6 X 100 em). The column, equilibrated and eluted with phosphate-buffered saline, was run at 12 ml/hour with an ascending flow; 6-ml fractions were collected. The fractions, corresponding to each of the three obtained peaks, were pooled and concentrated by ultrafiltration (Amicon PM-IO; Amicon Corp., Lexington, Mass.) to the initial volume. 51er cytotoxicity test for humoral response.-Cell suspensions were prepared from lymphomas or normal thymuses by the tissues being minced in Hanks' balanced salt solution (HBSS). They were repeatedly washed and adjusted at 20x 106 living cells/ml in HBSS, and viability was determined by exclusion of trypan blue dye. The cells were then incubated with 200 ftCi Na2 51Cr0 4 /ml (Radiochemical Centre, Amersham, England) at 37° C for 45 minutes. The labeled cells were washed four times, adjusted to 1 X 106 cells/m!, and the mixture of 0.1 ml cells and 0.1 ml test serum at the proper dilution was incubated at 37° C for 30 minutes; three replicates were made for each sample. After incubation, 1 ml HBSS was added, the cells were sedimented by centrifugation, the supernatant was discarded, and 0.025 ml guinea pig complement (B.D. Merieux, Marseille, France), selected for absence of cytotoxicity on lymphoma cells and diluted 1:4, was added to two replicates, whereas the third one received 0.025 ml HBSS. The incubation was continued for 30 minutes longer. Then each tube was refilled with 2 ml HBSS, the cells were sedimented by centrifugation, and the radioactivity released in I ml of the supernatant was measured in a Packard Autogamma Scintillation Spectrometer (Packard Instrument Co., Inc., Downers Grove, Ill.) We obtained the maximum amount of releasable radioactivity by incubating, at 37° C for 60 minutes, three samples, each with 1 X 105 labeled cells in 2 ml distilled water, and measuring the radioactivity of 1 ml supernatant after centrifugation. The percentage of specific 51Cr release was calculated as follows: Experimental release-control release Maximum release-control release

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Received March 12, 1975; accepted June 17, 1975. Supported by a grant from the Consiglio Nazionale delle Ricerche, Rome, Italy. 3 Division of Experimental Oncology A, Istituto Nazionale per 10 Studio e la Cura dei Tumori, Via G. Venezian I, Milan 20133, Italy. 4 We thank Dr. G. Della Porta for constructive criticism and Ms. Piera Mondellini and Mr. Mario Azzini for excellent technical assist· ance. 1 2

JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO.4, OCTOBER 1975

945

946

PIEROTTI AND COLNAGHI

where the experimental release was the mean counts per minute (cpm) of the two replicates with complement, and the control was the radioactivity released after incubation with serum without complement. The control release for all tests had a mean value of 10.66±0.18% SE of the maximum. We controlled the complement cytotoxicity in each test by evaluating the 51Cr release of three samples of cells incubated in HBSS and complement and of three samples containing HBSS only; the mean value for all tests was 3.72 ± 0.49% SE of the maximum release.

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The two pools of serum from C3Hf and C57BL male mice bearing a syngeneic lymphosarcoma were tested on C3Hf and C57BL lymphoma cells. As depicted in textfigure I, the C3Hf serum exerted a high complement-dependent cytotoxic activity on C57LyUr24 target cells but not on syngeneic lymphoma cells, whereas the C57BL serum was ineffective on both types of target cells. To verify whether normal mice had the same behavior, we performed tests to determine if serum from C3Hf normal males was capable of lysing the C57LyUr24 cells. In addition, the serum of C3H/He and C3Hf normal virgin females was tested on the same target cell to control a possible sex-linked effect or an influence of the original foster nursing of C3Hf mice on C57BL mothers. The three sera exerted the same level of complement-dependent c,totoxicity on the lymphoma cells (text-fig. 2).

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Percent specific 6tCr release±sE G

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2.-Complement-dependent cytotoxicity on the C57LyUr24 target cell of serum from C3Hf normal males (0---0), C3Hf (e •••••), and C3H/He (D--O) normal females.

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The serum from C3Hf normal males was then tested at dilutions from 1; 1 to 1; 128 on various murine lymphomas induced in different ways and maintained in transplant in syngeneic C57BL, B6, or C3Hf mice. The results obtained with the undiluted serum, which gave the highest release in all instances, are reported in table 1. A strong, though variable, cytotoxic activity was observed on all chemically or radiation-induced C57BL and B6 lymphomas tested, whereas the Ed"G2 leukemia of B6 strain and the chemically induced lymphomas of the C3Hf strain were unaffected at all serum dilutions. Strain Dependence and Specificity of the Cytotoxicity of Normal Serum for Neoplastic Cells

Normal sera from C3Hf, AKR, CBA/l, BALB/c, DBAf2, and C57BL mice were tested at the same time on C57LyUr24 cells to compare their cytotoxic activity; in addition, 2 other lymphomas of C3Hf and BALB/c

947

NATURAL ANTIBODIES TO LYMPHOSARCOMA CELLS IN MICE TABLE

2.-Strain dependence of cytotoxic activity of normal mice sera and susceptibility of lymphoma cells

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strains were analyzed for their strain-dependent cell susceptibility to autologous and allogeneic sera. Various degrees of the natural response to C57BL lymphoma cells were found (table 2). Among the positive sera, the highest cytotoxicity was detected in C3Hf serum, whereas the CBA j J and AKR sera were cytotoxic at a lower level; BALBjc, DBAj2, and C57BL sera were negative. In the three strains in which both serum and lymphoma cells were tested, an inverse relationship was detected between the cytotoxicity of serum and the susceptibility of lymphoma cells. In fact, we found that C3Hf, AKR, and CBAj J sera were cytotoxic for lymphoma cells of C57BL and BALBjc strains, the sera of which were negative on all target cells examined. Although the serum of C3Hf mice was positive on susceptible cells, their lymphoma cells were negative with the autologous serum. This lack of reaction was confirmed with the allogeneic AKR and CBAIJ sera. The B6 strain had the same behavior as did the C57BL mice to serum and target cells (data not shown). 60

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SERUM DILUTION 3.-Complement-dependent cytotoxicity of C3Hf serum on C57BL (0---0) and BALBjc (0--0) lymphoma cells or on C57BL (••••• e) and BALBjc ~ normal adult thymus cells.

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4.-Complement-dependent cytotoxicity on EL 4 cells of normal C3Hf serum unabsorbed (--) or absorbed with B6 normal thymus (0------0), C3LyUrll (0--0), or EL 4 lymphoma cells